Protein kinases are a class of enzymes that catalyze the transfer of a phosphate group from ATP to a tyrosine, serine, threonine, or histidine residue located on a protein substrate. Protein kinases clearly play a role in normal cell growth. Many of the growth factor receptor proteins have intracellular domains that function as protein kinases and it is through this function that they effect signaling. The interaction of growth factors with their receptors is a necessary event in the normal regulation of cell growth, and the phosphorylation state of substrate proteins often is related to the modulation of cell growth.
It is widely known that abnormal protein phosphorylation may be directly linked to certain disease states or may be a contributing factor in the onset of such diseases. As a result, protein kinases have become the targets of new pharmaceutical research (Cohen, P. Nature Reviews Drug Discovery, 1:309-315, 2002). Various protein kinase inhibitors have been used clinically in the treatment of a wide variety of diseases, such as cancer, chronic inflammatory diseases, diabetes and stroke.
The protein kinases are a large and diverse family of enzymes that catalyze protein phosphorylation and play a key role in cellular signaling. Protein kinases may exert positive or negative regulatory effects, depending upon their target protein. Protein kinases are involved in specific signaling pathways which regulate cell functions such as, but not limited to, metabolism, cell cycle progression, cell adhesion, vascular function, apoptosis, and angiogenesis. As a result, malfunctions of cellular signaling have been associated with many diseases, the most characterized of which include cancer and diabetes. The regulation of signal transduction by cytokines and the association of signal molecules with proto-oncogenes and tumor suppressor genes have been well documented. Similarly, the connection between diabetes, viral infections and the conditions related thereto has also been associated with the regulation of protein kinases.
Because protein kinases regulate nearly every cellular process, including metabolism, cell proliferation, cell differentiation, and cell survival, they are attractive targets for therapeutic intervention for various disease states. For example, cell-cycle control and angiogenesis, in which protein kinases play a pivotal role are cellular processes associated with numerous disease conditions such as, but not limited to, cancer, inflammatory diseases, abnormal angiogenesis and diseases related thereto, atherosclerosis, macular degeneration, diabetes, obesity, and pain.
The elucidation of the intricacy of protein kinase pathways and the complexity of the relationship and interaction among and between the various protein kinases and kinase pathways highlights the importance of developing pharmaceutical agents capable of acting as protein kinase modulators, regulators or inhibitors that have beneficial activity on multiple kinases or multiple kinase pathways.
It has therefore been suggested that due to the complexity of intracellular signaling cascades of protein kinase pathways, agents that affect multiple pathways simultaneously may be required for meaningful clinical activity. Although it has been suggested that a single agent that provides combinatorial effects is an attractive notion, there is a need to identify and use single agents that target the right combination of multiple pathways that are clinically effective in a particular disease setting.
Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase encoded by two isoforms, GSK-3α and GSK-3β with molecular weights of 51 and 47 kDa, respectively. These share 97% sequence similarity in their kinase catalytic domains. The GSK-3α isoform has an extended glycine-rich N-terminal tail. A minor splice variant of GSK-3β has been identified (expressed at ˜15% of total) with a 13 amino acid insert within the kinase domain. This variant had a reduced activity towards tau. GSK-3 is highly conserved throughout evolution, and found in all mammals thus far with high homology in the kinase domain. Both isoforms are ubiquitously expressed in mammalian tissues, including the brain. Pharmacological GSK-3 inhibitors are not able to selectively inhibit one of the isoforms.
GSK-3β plays an important role in the control of metabolism, differentiation and survival. It was initially identified as an enzyme able to phosphorylate and hence inhibit glycogen synthase. Subsequently, it was recognized that GSK-3β was identical to tau protein kinase 1 (TPK1), an enzyme that phosphorylates tau protein in epitopes that are also found to be hyperphosphorylated in Alzheimer's disease and in several taupathies.
Interestingly, protein kinase B (AKT) phosphorylation of GSK-3β results in a loss of kinase activity, and it has been proposed that this inhibition may mediate some of the effects of neurotrophic factors. Moreover, phosphorylation of β-catenin (a protein involved in cell survival) by GSK-3β, results in its degradation by an ubiquitinilation dependent proteasome pathway.
Therefore it appears that inhibition of GSK-3β activity may result in neurotrophic activity. There is evidence that lithium, an uncompetitive inhibitor of GSK-3β, enhances neuritogenesis in some models and can also increase neuronal survival, through the induction of survival factors such as Bcl-2 and the inhibition of the expression of proapoptotic factors such as P53 and Bax.
Further studies have shown that β-amyloid increases GSK-3β activity and tau protein phosphorylation. Moreover, this hyperphosphorylation as well as the neurotoxic effects of β-amyloid are blocked by lithium chloride and by a GSK-3β antisense mRNA. These observations taken together suggest that GSK-3β may be the link between the two major pathological processes in Alzheimer's disease: abnormal APP (Amyloid Precursor Protein) processing and tau protein hyperphosphorylation.
These experimental observations indicate that GSK-3β may find application in the prevention and treatment of the neuropathological consequences and the cognitive and attention deficits associated with Alzheimer's disease, as well as other acute and chronic neurodegenerative diseases. These include, but are not limited to: Parkinson's disease, tauopathies (e.g. frontotemporoparietal dementia, corticobasal degeneration, Pick's disease, progressive supranuclear palsy) and other dementia including vascular dementia; acute stroke and other traumatic injuries; cerebrovascular accidents (e.g. age related macular degeneration); brain and spinal cord trauma; peripheral neuropathies; retinopathies and glaucoma.
GSK-3β may also have utility in the treatment of other diseases such as: non-insulin dependent diabetes and obesity; manic depressive illness; schizophrenia; alopecia; inflammation; cancers such as breast cancer, non-small cell lung carcinoma, thyroid cancer, T or B-cell leukemia and several virus-induced tumors.
Rho kinases (ROCKs), the first Rho effectors to be described, are serine/threonine kinases that are important in fundamental processes of cell migration, cell proliferation and cell survival. Abnormal activation of the Rho/ROCK pathway has been observed in various disorders. Examples of disease states in which compounds of the present invention have potentially beneficial therapeutic effects due to their anti vasospasm activity includes cardiovascular diseases such as hypertension, chronic and congestive heart failure, cardiac hypertrophy, restenosis, chronic renal failure, cerebral vasospasm after subarachnoid bleeding, pulmonary hypertension and atherosclerosis. The muscle relaxing property is also beneficial for treating asthma, male erectile dysfunctions, female sexual dysfunction, and over-active bladder syndrome. Injury to the adult vertebrate brain and spinal cord activates ROCKs, thereby inhibiting neurite growth and sprouting. Inhibition of ROCKs results in induction of new axonal growth, axonal rewiring across lesions within the CNS, accelerated regeneration and enhanced functional recovery after acute neuronal injury in mammals (spinal-cord injury, traumatic brain injury). Inhibition of the Rho/ROCK pathway has also proved to be efficacious in other animal models of neurodegeneration like stroke, inflammatory and demyelinating diseases, Alzheimer's disease as well as the treatment of pain. Rho/ROCK pathway inhibitors therefore have potential for preventing neurodegeneration and stimulating neuroregeneration in various neurological disorders, including spinal-cord injury, Alzheimer's disease, stroke, multiple sclerosis, amyotrophic lateral sclerosis, as well as the treatment of pain. ROCK inhibitors have been shown to possess anti-inflammatory properties. Thus, compounds of the invention can be used as treatment for neuroinflammatory diseases such as stroke, multiple sclerosis, Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, and inflammatory pain, as well as other inflammatory diseases such as rheumatoid arthritis, osteoarthritis, asthma, irritable bowel syndrome, Crohn's disease, psoriasis, ulcerative colitis, Lupus, and inflammatory bowel disease. Since ROCK inhibitors reduce cell proliferation and cell migration, they could be useful in treating cancer and tumor metastasis. Further more, there is evidence suggesting that ROCK inhibitors suppress cytoskeletal rearrangement upon virus invasion, thus they also have potential therapeutic value in anti-viral and anti-bacterial applications. ROCK inhibitors are also useful for the treatment of insulin resistance and diabetes. Further, ROCK inhibitors have been shown to ameliorate progression of cystic fibrosis (Abstract S02.3, 8th World Congress on Inflammation, Copenhagen, Denmark, Jun. 16-20, 2007).
In addition, Rho-associated coiled-coil forming protein kinases (ROCK)-1 and -2, have been shown to enhance myosin light chain (MLC) phosphorylation by inhibiting MLC phosphatase as well as phosphorylating MLC. This results in the regulation of actin-myosin contraction. Recent reports have demonstrated that inhibition of ROCK results in disruption of inflammatory cell chemotaxis as well as inhibition of smooth muscle contraction in models of pulmonary inflammation associated with asthma. Therefore, the inhibitors of the Rho/ROCK pathway should be useful for the treatment of asthma.
The Janus kinases (JAKs) are an important family of intracellular protein tyrosine kinases (PTKs), with 4 mammalian members, JAK1, JAK2, JAK3, and TYK2, as well as homologs in chicken, fish, and Drosophila. The JAKs play critical roles in several important intracellular signaling pathways, including the eponymous JAK/STAT pathway, central to the mediation of cytokine signaling. It is this pivotal role in cytokine signaling that underpins the notion that specific JAK inhibitors may be therapeutically deployed in situations where cytokine activity results in disease. Important examples of this include autoimmune diseases such as rheumatoid arthritis and psoriasis, myeloproliferative syndromes such as, leukemias, lymphomas, and cardiovascular diseases.
JAK2, a member of the Janus kinase (JAK) family of protein tyrosine kinases (PTKs), is an important intracellular mediator of cytokine signaling. Mutations of the JAK2 gene are associated with hematologic cancers, and aberrant JAK activity is also associated with a number of immune diseases, including rheumatoid arthritis.
Aurora kinases are a family of multigene mitotic serine-threonine kinases that functions as a class of novel oncogenes. These kinases comprise aurora-A, aurora-B, and aurora-B members. These are hyperactivated and/or over-expressed in several solid tumors including but not limited to breast, ovary, prostate, pancreas, and colorectal cancers. In particular aurora-A is a centrosome kinase, and its localization depends on the cell cycle and plays an important role cell cycle progression and cell proliferation. Aurora-A is located in the 20q13 chromosome region that is frequently amplified in several different types of malignant tumors such as colorectal, breast and bladder cancers. Inhibition of aurora kinase activity could help to reduce cell proliferation, tumor growth and potentially tumorigenesis.
Eukaryotic cells divide by a directed, step-wise process referred to as the cell cycle. Cells must first replicate their DNA in S phase before separating their sister chromatids in mitosis (karyokinesis) and splitting off into two daughter cells (cytokinesis). In mammalian cells, DNA replication must be initiated at multiple sites (replication origins) throughout the genome to ensure that all the genetic material is duplicated prior to mitosis. To maintain genome integrity, DNA must be replicated only once per cell cycle, and so this process is highly regulated and governed by checkpoints. Before replication is initiated, origins must be licensed through the formation of pre-replication complexes (pre-RCs) in early G1. Formation of pre-RCs involves the step-wise binding of the origin recognition complex (ORC) to origins followed by the binding of the loading factors Cdc6 and Cdt1. These proteins then recruit the putative DNA replicative helicase complex, MCM2-7. Once this pre-RC is formed, replication initiation requires the activation of S-phase-promoting serine/threonine kinases, Cyclin/Cdks and Cdc7/Dbf4. These kinases consist of an enzymatic subunit (CDKs and Cdc7) and a regulatory sub-unit (Cyclins for CDKs; Dbf4 or Drf1 for Cdc7). They phosphorylate multiple MCMs in pre-RCs in a sequential manner, thereby activating the helicase and recruiting other DNA replication factors (Cdc45, GINS complex, etc.) for DNA synthesis (for reviews, see Kim, J. M., et al. (2003). Functions of mammalian Cdc7 kinase in initiation/monitoring of DNA replication and development. Mutat. Res. 532, 29-40; Kim, J. M., et al. (2004). Genetic dissection of mammalian Cdc7 kinase: cell cycle and developmental roles. Cell Cycle 3, 300-304; Lau, E., et al. (2006). The functional role of Cdc6 in S-G2/M in mammalian cells. EMBO Rep. 7, 425-430; Lau, E., et al. (2007). The role of pre-replicative complex (pre-RC) components in oncogenesis. Faseb J. 21, 3786-3794; Stillman, B. (2005). Origin recognition and the chromosome cycle. FEBS Lett. 579, 877-884). MCM2 Serine-40 and Serine-53 are well-characterized phosphorylation sites for Cdc7/Dbf4 (Cho et al., 2006; Montagnoli et al., 2006; Tsuji et al., 2006).
Inhibiting regulators of replication initiation, such as Cdc6, Cdc7/Dbf4 or Cdc7/Drf1, has lethal consequences in cancerous cells, whereas normal cells are able to arrest and resume normal divisions once initiation activity is restored (Feng, D., et al. (2003). Inhibiting the expression of DNA replication-initiation proteins induces apoptosis in human cancer cells. Cancer Res. 63, 7356-7364; Montagnoli, A., et al. (2004). Cdc7 inhibition reveals a p53-dependent replication checkpoint that is defective in cancer cells. Cancer Res 64, 7110-7116; see Lau, E., et al. (2006). Is there a pre-RC checkpoint that cancer cells lack? Cell Cycle 5, 1602-1606, for review). Small molecule inhibitors of the protein kinase Cdc7 are thus attractive candidates for therapeutic intervention in cancer, inflammation and other cell proliferative disorders.
Accordingly, there remains a need for the development of methods comprising the use of a single agent drug capable of targeting specific sets of kinases or kinase pathways. In particular such methods affect the right combination of multiple targets thereby achieving clinical efficacy.